Method of immunoassaying a component to be measured

ABSTRACT

A method of immunoassaying a component to be measured in a sample containing hemoglobin, which comprises reacting a component to be measured in a sample containing hemoglobin with an antibody capable of binding to the component in the presence of a bile acid derivative different from a bile acid derivative that is inherently contained in the sample; a method of suppressing an interference of hemoglobin in immunoassaying a component to be measured in a sample containing hemoglobin, which comprises reacting a component to be measured in a sample containing hemoglobin with an antibody capable of binding to the component in the presence of a bile acid derivative different from a bile acid derivative that is inherently contained in the sample; a reagent of immunoassay of a component to be measured in a sample containing hemoglobin, which comprises a bile acid derivative, are described.

TECHNICAL FIELD

The present invention relates to a method of immunoassaying a componentto be measured in a sample containing hemoglobin, a reagent ofimmunoassay, and a method of suppressing an interference of hemoglobinin a method of immunoassaying.

BACKGROUND ART

In a method of immunoassaying a component to be measured in blood thatexists outside blood cells such as red and white blood cells, serum orplasma prepared by removing blood cells from whole blood is used as asample. However, since removal of blood cells requires specialequipments such as a centrifuge and is troublesome, methods ofmeasurement using whole blood as a sample have been proposed (see PatentDocument 1). When whole blood is used as a sample, it is problematicthat a measurement is affected by blood cell components such ashemoglobin or blood cell membrane components contaminated in the samplethrough hemolysis. These components affect the optical detection system,inhibit immunoreactions, and adsorb the substance to be measured. Thereare reports of methods of immunoassaying using whole blood as a sample,not accompanied by hemolysis to avoid such problems (see PatentDocuments 2, 3, 4, and 5).

When measuring components in blood cell such as intracellular proteins,blood cells must be lysed; therefore, the above-described methods whichdo not accompany hemolysis cannot be used. In such cases, methods whichcomprise separation of the blood cells of interest by flow cytometryfollowed by lysis of the isolated cells and measurement of the desiredcomponent in blood cell are used, but these methods require specialequipments for flow cytometry and are troublesome.

On the other hand, as an example of convenient methods for measuringintracellular proteins, a method of immunoassaying MxA protein using asa sample for measurement, lysates of blood cells in whole blood preparedusing a surfactant, has been reported (Non-patent Documents 1 and 2).

-   [Patent Document 1] Japanese Patent Application Kokai Publication    No. (JP-A) H10-48214 (unexamined, published Japanese patent    application)-   [Patent Document 2] JP-A (Kokai) H06-265554-   [Patent Document 3] WO 96/04558-   [Patent Document 4] WO 02/73203-   [Patent Document 5] JP-A (Kokai) 2004-45395-   [Non-patent Document 1] Journal of Interferon Research, (USA), 1992,    Vol. 12, No. 2, p. 67-74-   [Non-patent Document 2] Pediatric Research, (USA), 1997, Vol. 41,    No. 5, p. 647-650

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

An objective of the present invention is to provide a method and areagent of immunoassaying a component to be measured in a samplecontaining hemoglobin to suppress an interference of hemoglobin, and amethod of suppressing an interference of hemoglobin in a method ofimmunoassaying a component to be measured in a sample containinghemoglobin.

Means for Solving the Problems

The present inventors found that in a method of immunoassaying acomponent to be measured in a sample, the component can be accuratelymeasured by reacting the component with an antibody capable of bindingto the component in the presence of a bile acid derivative differentfrom a bile acid derivative that is inherently contained in the sample,and thereby completed the present invention. More specifically, thepresent invention relates to [1] to [24] below.

-   [1] A method of immunoassaying a component to be measured in a    sample containing hemoglobin, which comprises reacting a component    to be measured with an antibody capable of binding to the component    in the presence of a bile acid derivative different from a bile acid    derivative that is inherently contained in the sample.-   [2] The method according to [1], which comprises reacting a    component to be measured in a sample containing hemoglobin with an    antibody capable of binding to the component, further in the    presence of a polyoxyethylene nonionic surfactant.-   [3] The method according to [1] or [2], wherein the method of    immunoassay is a sandwich method or a competition method.-   [4] The method according to [1] or [2], wherein reacting the    component with an antibody capable of binding to the component is:-   (1) reacting the component with a first antibody capable of binding    to the component and a labeled second antibody capable of binding to    the component,-   (2) reacting the component with a labeled competitive substance and    an antibody capable of binding to both of the component and the    competitive substance, or-   (3) reacting the component with a competitive substance and a    labeled antibody capable of binding to both of the component and the    competitive substance.-   [5] The method according to any one of [1] to [4], wherein the bile    acid derivative different from a bile acid derivative that is    inherently contained in the sample is a bile acid derivative having    amphoteric surfactant function.-   [6] The method of any one of [1] to [4], wherein the bile acid    derivative different from a bile acid derivative that is inherently    contained in the sample is a bile acid derivative having nonionic    surfactant function.-   [7] The method of [5], wherein the bile acid derivative having    amphoteric surfactant function is    3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate (hereinafter,    abbreviated as CHAPS) or    3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxypropanesulfonate    (hereinafter, abbreviated as CHAPSO).-   [8] The method according to [6], wherein the bile acid derivative    having nonionic surfactant function is    N,N-bis(3-D-gluconamidopropyl)cholamide (hereinafter abbreviated as    BIGCHAP) or N,N-bis(3-D-gluconamidopropyl)deoxycholamide    (hereinafter abbreviated as deoxy-BIGCHAP).-   [9] The method according to any one of [2] to [8], wherein the    polyoxyethylene nonionic surfactant is polyoxyethylene alkylphenyl    ether.-   [10] The method according to any one of [1] to [9], wherein the    sample is whole blood.-   [11] The method according to any one of [1] to [10], wherein the    component to be measured is MxA protein.-   [12] A reagent of immunoassay of a component to be measured in a    sample containing hemoglobin, which comprises a bile acid derivative    and a member selected from the group consisting of (1) to (3) below:-   (1) a first antibody capable of binding to the component and a    labeled second antibody capable of binding to the component;-   (2) a labeled competitive substance and an antibody capable of    binding to both of the component and the competitive substance; and-   (3) a competitive substance and a labeled antibody capable of    binding to both of the component and the competitive substance.-   [13] The reagent according to [12], which further comprises a    polyoxyethylene nonionic surfactant.-   [14] The reagent according to [12] or [13], wherein the bile acid    derivative is a bile acid derivative having amphoteric surfactant    function.-   [15] The reagent according to [12] or [13], wherein the bile acid    derivative is a bile acid derivative having nonionic surfactant    function.-   [16] The reagent according to [14], wherein the bile acid derivative    having amphoteric surfactant function is CHAPS or CHAPSO.-   [17] The reagent according to [15], wherein the bile acid derivative    having nonionic surfactant function is BIGCHAP or deoxy-BIGCHAP.-   [18] The reagent according to any one of [13] to [17], wherein the    polyoxyethylene nonionic surfactant is polyoxyethylene alkylphenyl    ether.-   [19] A method of suppressing an interference of hemoglobin in    immunoassaying a component to be measured in a sample containing    hemoglobin, which comprises reacting a component to be measured in a    sample containing hemoglobin with an antibody capable of binding to    the component in the presence of a bile acid derivative different    from a bile acid derivative that is inherently contained in the    sample.-   [20] The method according to [19], wherein the bile acid derivative    different from a bile acid derivative that is inherently contained    in the sample is a bile acid derivative having amphoteric surfactant    function.-   [21] The method according to [19], wherein the bile acid derivative    different from a bile acid derivative that is inherently contained    in the sample is a bile acid derivative having nonionic surfactant    function.-   [22] The method according to [20], wherein the bile acid derivative    having amphoteric surfactant function is CHAPS or CHAPSO.-   [23] The method according to [21], wherein the bile acid derivative    having non-ionic surfactant function is BIGCHAP or deoxy-BIGCHAP.-   [24] The method according to any one of [19] to [23], which    comprises reacting a component to be measured in a sample containing    hemoglobin with an antibody capable of binding to the component,    further in the presence of a polyoxyethylene nonionic surfactant.

Effects Of The Invention

The present invention provides a method of immunoassaying and a reagenttherefor that enable accurate measurement of a component to be measuredin a sample containing hemoglobin, and also provides a method ofsuppressing an interference of hemoglobin in a method of immunoassayinga component to be measured in a sample containing hemoglobin.

BEST MODE FOR CARRYING OUT THE INVENTION

(1) Sample Containing Hemoglobin

Examples of the sample containing hemoglobin used in the method ofimmunoassaying of the present invention include a sample containinghemoglobin and a sample suspected to contain hemoglobin. Examples of thesample containing hemoglobin and the sample suspected to containhemoglobin include whole blood, blood cell fraction containing red bloodcells prepared from whole blood, plasma or serum suspected of hemolysis,red blood cells, and arbitrary samples with added hemoglobin. As to thewhole blood, blood itself collected from a subject as well as bloodsubjected to treatment can be used, and is preferably blood subjected totreatment. Examples of such treatment include anticoagulation treatmentand hemolysis treatment, and these treatments may be used incombination.

In case the component to be measured is an intracellular component of ablood cell, the whole blood is preferably blood subjected to hemolysistreatment and is, in particular, preferably blood subjected to bothanticoagulation treatment and hemolysis treatment. Examples of theanticoagulation treatment include a treatment in which EDTA, heparin, orsuch is added to the collected blood. Examples of the hemolysistreatment include addition of a surfactant or saponin solution, mixingwith a hypotonic solution, freeze-thawing, and sonication.

(2) Component to be Measured

The component to be measured in the present invention is notparticularly limited as long as it is a component in a sample that maycontain hemoglobin, and examples of the component include a nucleicacid, a protein, a lipid, a vitamin, and a polysaccharide. Examples ofthe nucleic acid include DNA, RNA, ATP, ADP, AMP, and cyclic AMP.Examples of the protein include an enzyme, a hormone, and various typesof peptides.

The preferred component in the present invention includes a substancecontained within the cell, and a protein induced within cells by variouscytokines such as interferon.

A specific example of the component is MxA protein induced withincytoplasm by type-I interferon (Mol. Cell. Biol., 9, 5062-5072, 1989; J.Virol. 64, 1171-1181, 1990).

(3) Bile Acid Derivative

The bile acid derivative in the present invention is a bile acidderivative different from a bile acid derivative that is inherentlycontained in the sample. The bile acid derivative different from a bileacid derivative that is inherently contained in the sample is notparticularly limited, as long as it is a bile acid derivative thatenables the method of immunoassaying and the method of suppressing aninterference of hemoglobin of the present invention, and is preferably abile acid derivative having amphoteric surfactant function or nonionicsurfactant function.

Examples of the bile acid derivative having amphoteric surfactantfunction include 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate(hereinafter, abbreviated as CHAPS) and3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxypropanesulfonate(hereinafter, abbreviated as CHAPSO).

Examples of the bile acid derivative having nonionic surfactant functioninclude N,N-bis(3-D-gluconamidopropyl)cholamide (hereinafter,abbreviated as BIGCHAP) and N,N-bis(3-D-gluconamidopropyl)deoxycholamide(hereinafter, abbreviated as deoxy-BIGCHAP).

The bile acid derivative shows an effect of suppressing an interferenceof hemoglobin in the reaction of the component to be measured and anantibody capable of binding to the component, and in particular, CHAPS,CHAPSO, BIGCHAP, and deoxy-BIGCHAP are preferably used.

The bile acid derivative is used at a concentration in the range of 1-to 50-times the critical micelle concentration (cmc), and in particular,1- to 10-times the cmc concentration is preferred. In the presentinvention, the bile acid derivative can be used alone (one type), or incombination of two or more types of bile acid derivatives.

(4) Polyoxyethylene Nonionic Surfactant

Polyoxyethylene nonionic surfactant works to increase a measurementsensitivity in the method of immunoassaying of the present invention,and are preferably present during the immunoreaction.

Examples of the polyoxyethylene nonionic surfactant in the presentinvention include polyoxyethylene alkylphenyl ether, polyoxyethylenealkyl ether, and polyoxyethylene sorbitan fatty acid ester, and ispreferable polyoxyethylene alkylphenyl ether. Examples of the alkyl inthe polyoxyethylene alkylphenyl ether include octyl and nonyl. Aspecific example (commercially available product) of polyoxyethylenealkylphenyl ether is Nonidet P-40 (polyoxyethylene nonylphenyl ether).

In the present invention, the polyoxyethylene nonionic surfactant can beused alone (one type), or in combination of two or more types ofpolyoxyethylene nonionic surfactants. In the method of immunoassaying ofthe present invention, the concentration of the polyoxyethylene nonionicsurfactant is preferably 0.01% to 2.0%, more preferably 0.05% to 1.8%,and particularly preferably 0.1% to 1.4%.

(5) Antibody and Labeled Antibody

The antibody used in the method of immunoassaying of the presentinvention is not particularly limited, as long as it is an antibody thatspecifically binds to the component to be measured; and either apolyclonal antibody or a monoclonal antibody may be used, but amonoclonal antibody is preferred. Furthermore, the antibody to be usedin the present invention includes an antibody fragment. Specificexamples include an antibody fragment in which the Fc portion has beenremoved, such as Fab obtained by papain treatment of an antibody,F(ab′)₂ obtained by pepsin treatment, and Fab′ obtained by pepsintreatment and reduction treatment. In particular, the preferred antibodyfragment is F(ab′)₂.

The antibody to be used in the present invention can be obtained bystandard methods using the component to be measured or a part thereof asan antigen, and a commercially available product can be also be used.

In case the component to be measured is MxA protein, examples of theantibody that specifically binds to MxA protein include anti-human MxAprotein monoclonal antibodies KM1122, KM1123, KM1124, KM1125, KM1126,KM1127, KM1128, KM1129, KM1130, KM1131, KM1132, KM1133, KM1134, andKM1135 produced by hybridoma cell lines KM1122, KM1123, KM1124 (FERMBP-4729), KM1125, KM1126, KM1127, KM1128, KM1129, KM1130, KM1131, KM1132(FERM BP-4730), KM1133, KM1134, and KM1135 (FERM BP-4731), respectively,which are described in International Publication No. WO 96/05230.

The labeled antibody in the present invention is an antibody that may beused for the method of immunoassaying a component to be measured in thepresent invention, and is produced by a method, described later, usingthe antibody used in the present invention and a labeling substancedescribed below.

(6) Competitive Substance and Labeled Competitive Substance

In the present invention, the term “competitive substance” refers to asubstance that can bind to “an antibody capable of binding to acomponent to be measured” to be used in the method of immunoassaying ofthe present invention, and competes against the component for binding;and the component itself is also included. A “competitive substance” isused in a measurement of a component to be measured in a sample using acompetitive method. Therefore, the antibody capable of binding to acomponent to be measured used in the competitive method is an antibodycapable of binding to a component to be measured, a competitivesubstance, and a labeled competitive substance. While the antibody bindsto the component to form an immune complex, it also binds to thecompetitive substance to form an immune complex.

The competitive substance is preferably a substance structurallyidentical to an epitope recognized by the antibody capable of binding toa component to be measured. In addition, as to the ability of binding tothe antibody capable of binding to the component to be measured, thecompetitive substance has, preferably, a comparable level to thecomponent. As the competitive substance, the component to be measureditself is preferred.

The labeled competitive substance in the present invention is asubstance that can be used for the method of immunoassaying a componentto be measured of the present invention, and is produced by a methoddescribed later, using the above-described competitive substance and alabeling substance to be described later.

(7) Method of Immunoassaying

The method of immunoassaying of the present invention is an immunoassayof measuring a component to be measured in a sample containinghemoglobin, which comprises reacting the component in the samplecontaining hemoglobin with an antibody capable of binding to thecomponent in the presence of a bile acid derivative different from abile acid derivative that is inherently contained in the sample.

A ratio of the sample containing hemoglobin and a bile acid derivativedifferent from a bile acid derivative that is inherently contained inthe sample is preferably 1:1 to 1:1000, and in case the samplecontaining hemoglobin is whole blood, a ratio of 1:2 to 1:49 ispreferred, and a ratio of 1:4 to 1:9 is particularly preferred. Whenadding the bile acid derivative different from a bile acid derivativethat is inherently contained in the sample to the sample containinghemoglobin, the temperature during the addition is preferablyapproximately 2° C. to 40° C., and measurement is, preferably, carriedout within 24 hours after the addition.

The method of immunoassaying is not particularly limited as long as itis a method based on an antigen-antibody reaction, and there are norestrictions on a method of operation, a presence or absence of alabeling substance, a type of labeling substance, a carrier, and apresence or absence of B/F separation.

The antigen-antibody reaction may either be a competitive reactionmethod or a non-competitive reaction method.

The detection method may either be an unlabeling-method in which theresult of antigen-antibody reaction by agglutination or such is detecteddirectly, or a labeling-method in which the result of antigen-antibodyreaction is detected using a labeling substance, and the labeling methodis particularly preferred from the aspect of measurement sensitivity.

In the method of immunoassaying of the present invention, either aheterogeneous method that requires B/F separation, or a homogeneousmethod that does not require a B/F separation can be used.

As to a reaction phase, either a liquid phase method in which allreactions are performed in liquid phase, or a solid phase method inwhich reactions are performed in the state of one part of the reactantsin the immune reaction being immobilized to a solid phase, can be used.

Specific examples of the measurement method include the method describedin “Bio Kensayaku Kaihatsu Manual (Biological Diagnostic AgentDevelopment Manual)”, CMC; jointly-edited by Ishikawa, E. et al. “KosoMeneki Sokuteiho (Enzyme Immunoassay)” 3rd edition, Igaku-Shoin; andDocument, Nippon Rinsho (Japanese Journal of Clinical Medicine), Vol.53, No. 9.

The methods of Measurement Methods 1 to 4 are shown below as specificexamples of the method of immunoassaying of the present invention, butthe present invention is not limited thereto. Measurement Method 1 is asandwich method which is a non-competitive reaction method, MeasurementMethods 2 and 3 are competitive methods in which a component to bemeasured in a sample competes with a competitive substance, andMeasurement Method 4 is a homogeneous method in which separation of theimmune complex from labeled antibody or labeled competitive substancewhich is not contained in the immune complex (B/F separation) is notcarried out.

Measurement Method 1

A measurement method in which steps (a) to (e) below are carried outsequentially:

-   (a) reacting a component to be measured in a sample containing    hemoglobin with a first antibody that binds specifically to the    component, in the presence of a bile acid derivative different from    a bile acid derivative that is inherently contained in the sample,    or in the presence of a bile acid derivative different from a bile    acid derivative that is inherently contained in the sample and a    polyoxyethylene nonionic surfactant to form an immune complex of the    first antibody and the component;-   (b) reacting the immune complex produced in step (a) with a labeled    second antibody capable of binding to the component, in the presence    of a bile acid derivative different from a bile acid derivative that    is inherently contained in the sample, or in the presence of a bile    acid derivative different from a bile acid derivative that is    inherently contained in the sample and a polyoxyethylene nonionic    surfactant, to form an immune complex of the first antibody, the    component, and the labeled antibody;-   (c) separating the immune complex formed in step (b) from the    labeled antibody which is not contained in the immune complex;-   (d) measuring the amount of label in the immune complex produced in    step (b); and-   (e) determining the concentration of the component in the sample on    the basis of the amount of label in the immune complex measured in    step (d).

The first antibody is preferably immobilized on an insoluble carrier.Step (a) and step (b) may be performed sequentially or simultaneously.As long as the second antibody can bind to the component bound to thefirst antibody, the site of the component recognized by the firstantibody may be the same as or different from the site of the componentrecognized by the second antibody, and these sites are preferablydifferent. Furthermore, as described later, in step (e), theconcentration of the component in the sample can be determined by usinga calibration curve that shows the relationship between theconcentrations of the component and the measured values (amount ofinformation originated from label) prepared in advance using thecomponents with known concentrations.

Measurement Method 2

A measurement method in which steps (a) to (d) below are carried outsequentially:

-   (a) reacting a component to be measured in a sample containing    hemoglobin and a labeled competitive substance with an antibody    capable of binding to both of the component and the labeled    competitive substance, in the presence of a bile acid derivative    different from a bile acid derivative that is inherently contained    in the sample or in the presence of a bile acid derivative different    from a bile acid derivative that is inherently contained in the    sample and a polyoxyethylene nonionic surfactant, to form an immune    complex of the antibody and the labeled competitive substance, and    an immune complex of the antibody and the component;-   (b) separating the immune complex of the antibody and the labeled    competitive substance from unreacted labeled competitive substance;-   (c) measuring the amount of label in the immune complex of the    antibody and the labeled competitive substance formed in step (a);    and-   (d) determining the concentration of the component in the sample on    the basis of the amount of label in the immune complex measured in    step (c).

The antibody is preferably immobilized on an insoluble carrier.Furthermore, as described later, in step (d), the concentration of thecomponent in the sample can be determined by using a calibration curvethat shows the relationship between the concentration of the componentand the measured values (amount of information originated from label)prepared in advance using the component with known concentrations.

Measurement Method 3

A measurement method in which steps (a) to (d) below are carried outsequentially:

-   (a) reacting a component to be measured in a sample containing    hemoglobin and a competitive substance with a labeled antibody    produced by binding a label to an antibody capable of binding to    both of the component and competitive substance, in the presence of    a bile acid derivative different from a bile acid derivative that is    inherently contained in the sample, or in the presence of a bile    acid derivative different from a bile acid derivative that is    inherently contained in the sample and a polyoxyethylene nonionic    surfactant, to form an immune complex of the labeled antibody and    the competitive substance, and an immune complex of the labeled    antibody and the component;-   (b) separating the immune complex of the labeled antibody and the    competitive substance from unreacted labeled antibody and the immune    complex of the labeled antibody and the component;-   (c) measuring the amount of label in the immune complex of the    labeled antibody and competitive substance; and-   (d) determining the concentration of the component in the sample on    the basis of the amount of label in the immune complex measured in    step (c).

The competitive substance is preferably immobilized on an insolublecarrier. In case the competitive substance has the same structure as thecomponent, a competitive substance immobilized on an insoluble carrieris used in step (a). Furthermore, as described later, in step (d), theconcentration of the component in the sample can be determined by usinga calibration curve that shows the relationship between theconcentrations of the component and the measured values (amount ofinformation originated from label) prepared in advance using thecomponents with known concentrations.

Measurement Method 4

A measurement method comprising steps (a) to (c) below:

-   (a) reacting a component to be measured in a sample containing    hemoglobin with labeled antibody 1 in which a first antibody capable    of binding to the component is labeled with labeling substance 1,    and with labeled antibody 2 in which a second antibody capable of    binding to the component is labeled with labeling substance 2,    different from labeling substance 1, in the presence of a bile acid    derivative different from a bile acid derivative that is inherently    contained in the sample, or in the presence of a bile acid    derivative different from a bile acid derivative that is inherently    contained in the sample and a polyoxyethylene nonionic surfactant,    to form an immune complex of labeled antibody 1, the component, and    labeled antibody 2;-   (b) measuring the amount of change in the interaction between    labeling substance 1 and labeling substance 2 in the immune complex    formed in step (a); and-   (c) determining the amount of the component in the sample on the    basis of the amount of change of interaction measured in step (b).

As long as the second antibody can bind to the component bound to thefirst antibody, the site of the component recognized by the firstantibody may be the same as or different from the site of the componentrecognized by the second antibody, and these sites are preferablydifferent. Furthermore, as described later, in step (c), theconcentration of the component in the sample can be determined by usinga calibration curve that shows the relationship between theconcentrations of the component and the measured values (amount ofinformation originated from label) prepared in advance using thecomponents with known concentrations.

As described above, Measurement Methods 1 to 3 are heterogeneous methodsthat involve B/F separation. Step (c) of Measurement Method 1 and step(b) of Measurement Methods 2 and 3 are steps of B/F separation. In casean antibody or a competitive substance is immobilized on an insolublecarrier, B/F separation can be performed easily by removing the reactionsolution and then washing the insoluble carrier. More specifically, byremoving the reaction solution after the antigen-antibody reaction, andwashing the insoluble carrier with a washing solution, the immunecomplexes formed on the insoluble carrier can be separated fromunreacted labeled substances (labeled antibody and labeled competitivesubstance).

Examples of the washing solution include phosphate buffered saline (pH7.2, 10 mmol/L phosphate buffer containing 0.15 mol/L sodium chloride;hereinafter referred to as PBS), PBS containing a surfactant, and anaqueous medium described later. Examples of the surfactant include anonionic surfactant such as Tween 20.

Furthermore, in Measurement Method 1, when the first antibody isimmobilized on an insoluble carrier, a step of washing the insolublecarrier can be inserted between step (a) and step (b) to removeunreacted reactants. In this case, step (b) becomes the step of reactingthe immune complex formed in step (a) with a labeled second antibodycapable of binding to the component, to form an immune complex of thefirst antibody, the component, and the labeled antibody.

In Measurement Method 2, in case the antibody capable of binding to thecomponent is not immobilized on an insoluble carrier, in step (c), aninsoluble carrier immobilized with a binding substance incapable ofbinding to the labeled competitive substance and capable of binding tothe antibody is allowed to react with the immune complexes to give theimmune complex bound to the insoluble carrier. After the reactionsolution is removed, the insoluble carrier is washed to separate theimmune complexes from the labeled competitive substance which is notcontained in the immune complex. Furthermore, in the presence of aninsoluble carrier immobilized with a binding substance incapable ofbinding to the labeled competitive substance and capable of binding tothe antibody, a reaction of formation of the immune complexes of step(a) is carried out to give a formation of the immune complexes and animmobilization of the immune complexes to the insoluble carriersimultaneously, and removal of the reaction solution followed by washingof the insoluble carrier lead to separation of the immune complexes fromlabeled competitive substance which is not contained in the immunecomplex. Examples of the binding substance incapable of binding to thelabeled competitive substance and capable of binding to the antibodyinclude an antibody capable of binding to the constant region of theantibody. In case the component is not a protein, a protein precipitantsuch as ammonium sulfate or polyethylene glycol can be added in step (c)to precipitate only the immune complex. After centrifugation of thereaction mixture, the immune complex can be separated from labeledcompetitive substance which is not contained in the immune complex.

In Measurement Method 1, in case the first antibody is not immobilizedon an insoluble carrier, separation of the labeled antibody contained inthe immune complex from the labeled antibody which is not contained inthe immune complex can be carried out by addition of an insolublecarrier immobilized with a binding substance incapable of binding to thelabeled antibody and capable of binding to the first antibody in thestep of B/F separation, followed by removal of a reaction solution andwashing of the insoluble carrier. Furthermore, separation of the labeledantibody contained in the immune complex from the labeled antibody whichis not contained in the immune complex can be carried out by formationof the immune complexes in the presence of an insoluble carrierimmobilized with a binding substance incapable of binding to the labeledantibody and capable of binding to the first antibody in the step offormation of the immune complexes, followed by removal of a reactionsolution and washing of the insoluble carrier. Examples of the bindingsubstance incapable of binding to the labeled antibody and capable ofbinding to the first antibody include an antibody against immunoglobulinof animal species used to produce the first antibody, in case the animalspecies used to produce the first antibody is different from the animalspecies used to produce the antibody (second antibody) used for thelabeled antibody; and an antibody capable of specifically binding to theconstant region of the first antibody, in case the first antibody is anantibody with constant region and the labeled antibody is an antibodyfragment such as Fab or F(ab′)₂, or Fab′ that does not have constantregion.

(8) Insoluble Carrier

The insoluble carrier for immobilizing an antibody or a competitivesubstance is not restricted as long as it can stably hold the antibodyor the competitive substance. Examples of the preferred material for theinsoluble carrier include a polymer material such as polystyrene,polycarbonate, polyvinyl toluene, polypropylene, polyethylene, polyvinylchloride, nylon, polymethacrylate, gelatin, agarose, cellulose,nitrocellulose, cellulose acetate, cellulose acetate, and polyethyleneterephthalate, glass, ceramics, magnetic particle, and metal. Examplesof the preferred form of insoluble carrier include tube, bead, plate,microparticle such as latex, and stick. For example, a polystyrenemicrotiter plate having 96 wells per plate is preferred.

(9) Immobilization of an Antibody or a Competitive Substance to anInsoluble Carrier

Examples of the method of immobilizing an antibody or a competitivesubstance to an insoluble carrier include a known method such as amethod using a physical bond, a method using a chemical bond, or acombination thereof. Examples of the physical bond include anelectrostatic bond, a hydrogen bond, and a hydrophobic bond. Examples ofthe chemical bond include a covalent bond and a coordinate bond. In caseusing a polystyrene microtiter plate for the method of immunoassay as aninsoluble carrier, a method of immobilization is exemplified thataddition of a solution of an antibody or a competitive substance to thewells of the plate is followed by incubation for one hour to one day at4° C. to 30° C. for physical adsorption.

The antibody or the competitive substance can be immobilized directly orindirectly on an insoluble carrier. Examples of the indirectimmobilization include a method comprising adding a biotinylatedantibody or a biotinylated competitive substance to an insoluble carrierimmobilized with avidin, and immobilizing the antibody or thecompetitive substance to the insoluble carrier through specificallybinding between biotin and avidin. Furthermore, an antibody capable ofspecifically binding to the antibody or an antibody capable ofspecifically binding to the competitive substance can be immobilized onthe insoluble carrier, and the antibody or the competitive substance canbe immobilized on the insoluble carrier through such an antibody.Alternatively, the antibody or the competitive substance may beimmobilized on the insoluble carrier by covalent bonds via a linker.

The linker is not restricted as long as it can form a covalent bondbetween both a functional group of the antibody or the component and afunctional group of the side chain of the insoluble carrier. In apreferred embodiment, for example, it is a molecule that concurrentlyhas a first reactive group that can react with a functional group of theantibody or the component, and a second reactive group that can reactwith a functional group of the side chain of the insoluble carrier.Preferably, the first reactive group is different from the secondreactive group. Examples of the functional group of the antibody or thecompetitive substance, and the functional group that the insolublecarrier has on its surface, include carboxy group, amino group, glycidylgroup, sulfhydryl group, hydroxy group, amide group, imino group,N-hydroxysuccinyl group, and maleimide group. Examples of the reactivegroup on the linker include groups such as arylazide, carbodiimide,hydrazide, aldehyde, hydroxymethyl phosphine, imide ester, isocyanate,maleimide, N-hydroxy succinimide (NHS) ester, pentafluorophenyl (PFP)ester, psoralen, pyridyl disulfide, and vinyl sulfone.

(10) Labeling of an Antibody or a Component to be Measured

Examples of the labeling substance for labeling an antibody or acomponent to be measured include an enzyme, a fluorescent substance, aluminescent substance, a radioisotope, biotin, digoxigenin, apolypeptide containing a tag sequence, a metallic colloid particle, anda colored latex particle.

Examples of the enzyme include alkaline phosphatase, peroxidase,galactosidase, glucuronidase, and luciferase.

Examples of the fluorescent substance include fluorescein isothiocyanate(FITC) and rhodamine B-isothiocyanate (RITC). Examples of the otherfluorescent substance include quantum dot (Science, 281, 2016-2018,1998), phycobiliprotein such as phycoerythrin, and afluorescence-emitting protein such as green fluorescent protein (GFP),red fluorescent protein (RFP), yellow fluorescent protein (YFP), andblue fluorescent protein (BFP).

Examples of the luminescent substance include acridinium and aderivative thereof, a ruthenium complex compound, and lophine. As to theruthenium complex compound, the compound that electrochemically emitslight with electron donors (described in Clin. Chem. 37, 9, 1534-1539,1991) is preferred.

Examples of the radioisotope include ³H, ¹⁴C, ³⁵S, ³²P, ¹²⁵I, and ¹³¹I.

Examples of the polypeptide containing a tag sequence include the FLAGpeptide (FLAG tag, Asp Tyr Lys Asp Asp Asp Asp Lys), polyhistidine (Histag, His His His His His His), myc epitope peptide (myc tag, Glu Gln LysLeu Ile Ser Glu Glu Asp Leu), and hemagglutinin epitope peptide (HA tag,Tyr Pro Tyr Asp Val Pro Asp Tyr Ala).

Labeling of the antibody or the component to be measured can be carriedout by a reaction that forms a covalent bond between the functionalgroup of the antibody or the component and the functional group of thelabeling substance, either with or without a linker. Examples of thefunctional group include a carboxyl group, an amino group, a glycidylgroup, a sulfhydryl group, a hydroxy group, an amido group, an iminogroup, a hydroxysuccinyl ester group, a maleimide group, and anisothiocyanate group. A condensation reaction between these functionalgroups can be performed.

Examples of the method of formation of bond without a linker include amethod using a carbodiimide compound such as EDC. In this case, anactive ester such as NHS or its derivatives can be used. Thecondensation reaction between an isothiocyanate group and an amino groupis preferred because it does not require other reagents, and proceedssimply by mixing under neutral to weakly alkaline conditions.

The linker is not restricted, as long as it can make a bond between thelabeling substance and the antibody via their respective functionalgroups. In a preferred embodiment, for example, the linker is a moleculethat has within the same molecule a first functional group that canreact with an amino acid residue of the antibody, and a secondfunctional group that can react with a functional group of the sidechain of the labeling substance. Preferably, the first functional groupis different from the second functional group. Examples of thefunctional group of the linker include the functional groups describedabove.

Examples of the method of making a radioisotope bond chemically includethe method described in Antibody Immunoconj. Radiopharm., 3, 60, 1990.

In case the labeling substance is an enzyme, avidin, afluorescence-emitting protein, a phycobiliprotein, or a polypeptide suchas a polypeptide comprising a tag sequence, the labeled antibody can beproduced as follows: producing an expression vector containing a DNAthat encodes a fusion protein of the labeling substance and theantibody, introducing the expression vector into a suitable host, andculturing the host (Molecular Cloning: A Laboratory Manual, 3rd Edition,Cold Spring Harbor Laboratory Press, 2001). A DNA encoding the fusionprotein can be obtained by cloning using PCR or the like of DNAs thatencode individually the antibody and the labeling substance, and linkingeach of the DNAs by a ligase reaction.

Examples of the labeling substances 1 and 2 used in the homogeneousmethod described in Measurement Method 4 of the above-mentioned (6)include labeling substances that initiate an interaction by binding to acomponent to be measured and coming near thereby. Examples of suchlabeling substances include fluorescent substances that exhibitfluorescence resonance energy transfer (FRET). FRET is a phenomenon thata fluorescent energy produced when the first fluorescent substance issubjected to excitation light is used as a fluorescent energy of thesecond fluorescent substance near the first fluorescent substance, andtakes place when the two kinds of fluorescent substances come near eachother to a distance of 1 to 10 nm. Examples of the combination of thefluorescent substances that exhibit FRET include a combination in whichthe fluorescent wavelength spectrum of one of the substances has someoverlap with the excitation wavelength spectrum of the other substance.Examples of the fluorescent substance include a fluorescent protein, alow-molecular-weight organic fluorescent dye, and an inorganic compound.Examples of the combination of fluorescent proteins that exhibit FRETinclude the combination of YFP [yellow mutant of green fluorescentprotein (GFP)] and CFP [cyan mutant of green fluorescent protein (GFP)].Examples of the combination of low-molecular-weight organic fluorescentdye include a combination of Cy3 and Cy5. Examples of the inorganiccompound include quantum dot (Science, 281, 2016-2018, 1998).

Furthermore, examples of the combination of the labeling substances inthe homogeneous method include a combination of achemiluminescence-producing enzyme and a fluorescent substance thatexhibit bioluminescence resonance energy transfer (BRET). Examples ofthe combination of an enzyme and a fluorescent substance that exhibitBRET include a combination that affords an overlap between theluminescence wavelength spectrum formed when the enzyme degrades itssubstrate and the excitation wavelength spectrum of the fluorescentsubstance. Examples of the combination include a combination of Renillaluciferase (Rluc) as the enzyme, Deep Blue C (manufactured by PackardBioScience) or such as the substrate, and GFP as the fluorescentsubstance. In this case, a light having a wavelength of 395 nm isproduced by degradation of the substrate by Rluc; and as GFP comes nearRluc, GFP receives the energy of this light and emits fluorescence atwavelength 510 nm which can be detected.

Examples of the combination of the labeling substances in thehomogeneous method include a combination of substances in which enzymeactivity appears when labeling substance 1 and labeling substance 2 comenear each other and bind in a certain orientation. Examples of thecombination of the labeling substances include a combination of the Δαsubunit of β-galactosidase as labeling substance 1 and the Δω subunit ofβ-galactosidase as labeling substance 2, and a combination of theN-terminal domain of Rluc as labeling substance 1 and the C-terminaldomain of Rluc as labeling substance 2.

(11) Antigen-antibody Reaction

An antigen-antibody reaction is preferably performed in an aqueousmedium. The reaction temperature is, for example, 0° C. to 50° C., andis preferably 4° C. to 40° C. The reaction time is preferably 5 minutesto 20 hours.

(12) Measurement of the Amount of Label

A suitable method of measuring the amount of label in the immune complexcan be selected according to the labeling substance. More specifically,in case the labeling substance is a coloring substance which is asubstance that absorbs light of a certain wavelength or the amount ofchange in turbidity (absorbance) caused by agglutination or the like ismeasured, a spectrophotometer, a multi-well plate reader, or such can beused. In case the labeling substance is a fluorescent substance, aspectrofluorometer, fluorescence multi-well plate reader, or such may beused. When the labeling substance is a luminescent substance, aluminescence photometer, luminescence multi-well plate reader, or suchcan be used. In case the labeling substance is a radioisotope, theamount of radioisotope can be determined by measuring the radioactivityusing a scintillation counter, a γ-well counter, or such.

In case the label is an enzyme, the amount of the label can bedetermined by measuring enzyme activity. For example, the amount of thelabel can be determined by reacting a substrate of the enzyme with theenzyme and measuring the substance formed.

In case the enzyme is peroxidase, peroxidase activity can be measured,for example, by a spectrophotometry, a fluorescence spectrophotometry,or such. Examples of the method of measuring peroxidase activity by aspectrophotometry include a method comprising reacting of peroxidasewith a combination of hydrogen peroxide and an oxidative coloringchromogen, which are the substrates of peroxidase, and measuring theabsorbance of the reaction solution using a spectrophotometer ormulti-well plate reader. Examples of the oxidative coloring chromogeninclude a leuco-type chromogen and an oxidative coupling-coloringchromogen.

The leuco-type chromogen is a substance that is converted into a dye byitself in the presence of hydrogen peroxide and a peroxidative substancesuch as peroxidase. Specific examples include tetramethylbenzidine,o-phenylenediamine,10-N-carboxymethylcarbamoyl-3,7-bis(dimethylamino)-10H-phenothiazine(CCAP), 10-N-methylcarbamoyl-3,7-bis(dimethylamino)-10H-phenothiazine(MCDP),N-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)diphenylaminesodium salt (DA-64), 4,4′-bis(dimethylamino)diphenylamine, andbis[3-bis(4-chlorophenyl)methyl-4-dimethylaminophenyl]amine (BCMA).

The oxidative coupling-coloring chromogen is a substance that forms adye by oxidative coupling of two compounds in the presence of hydrogenperoxide and a peroxidative substance such as peroxidase. Examples ofthe combination of two compounds include a combination of a coupler andan aniline compound (Trinder reagent), and a combination of a couplerand a phenol compound. Examples of the coupler include 4-aminoantipyrine(4-AA) and 3-methyl-2-benzothiazolinonehydrazine. Examples of theaniline compound include N-(3-sulfopropyl)aniline,N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (TOOS),N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline (MAOS),N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (DAOS),N-ethyl-N-(3-sulfopropyl)-3-methylaniline (TOPS),N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (HDAOS),N,N-dimethyl-3-methylaniline,N,N-bis(3-sulfopropyl)-3,5-dimethoxyaniline,N-ethyl-N-(3-sulfopropyl)-3-methoxyaniline,N-ethyl-N-(3-sulfopropyl)aniline,N-ethyl-N-(3-sulfopropyl)-3,5-dimethoxyaniline,N-(3-sulfopropyl)-3,5-dimethoxyaniline,N-ethyl-N-(3-sulfopropyl)-3,5-dimethylaniline,N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline,N-ethyl-N-(2-hydroxy-3-sulfopropyl)aniline,N-ethyl-N-(3-methylphenyl)-N′-succinylethylenediamine (EMSE),N-ethyl-N-(3-methylphenyl)-N′-acetylethylenediamine, andN-ethyl-N-(2-hydroxy-3-sulfopropyl)-4-fluoro-3,5-dimethoxyaniline(F-DAOS). Examples of the phenol compound include phenol,4-chlorophenol, 3-methylphenol, and 3-hydroxy-2,4,6-triiodobenzoic acid(HTIB).

Examples of the method of measuring the peroxidase activity by afluorescence spectrometry include a method comprising reacting ofperoxidase with a combination of hydrogen peroxide and a fluorescentsubstance, which are the substrates of peroxidase, and measuring theintensity of the generated fluorescence using a spectrofluorometer,fluorescence multi-well plate reader, or the like. Examples of thefluorescent substance include 4-hydroxyphenylacetic acid,3-(4-hydroxyphenyl)propionic acid, and coumarin.

Examples of the method of measuring the peroxidase activity by aluminescent measurement include a method comprising reacting ofperoxidase with a combination of hydrogen peroxide and a luminescentsubstance, which are the substrates of peroxidase, and measuring theintensity of the generated luminescence using a luminescence intensitymeter, luminescence multi-well plate reader, or the like. Examples ofthe luminescent substance include a luminol compound and a lucigenincompound.

In case the enzyme is alkaline phosphatase, the alkaline phosphataseactivity can be measured by, for example, a luminescent measurement.Examples of the method of measuring the alkaline phosphatase activity bya luminescent measurement include a method comprising reacting ofalkaline phosphatase with its substrate, and measuring the luminescenceintensity of the generated luminescence using a luminescence intensitymeter, luminescence multi-well plate reader, or the like. Examples ofthe substrate of alkaline phosphatase include3-(2′-spiroadamantane)-4-methoxy-4-(3′-phosphoryloxy)phenyl-1,2-dioxetanedisodium salt (AMPPD),2-chloro-5-{4-methoxyspiro[1,2-dioxetane-3,2′-(5′-chloro)tricyclo[3.3.1.13,7]decane]-4-yl}phenylphosphatedisodium salt (CDP-Star™),3-{4-methoxyspiro[1,2-dioxetane-3,2′-(5′-chloro)tricyclo[3.3.1.13,7]decane]-4-yl}phenylphosphatedisodium salt (CSPD™), and[10-methyl-9(10H)-acridinylidene]phenoxymethylphosphate disodium salt(Lumigen™ APS-5).

In case the enzyme is β-D-galactosidase, the β-D-galactosidase activitycan be measured by, for example, a spectrophotometry (colorimetricmethod), a luminescent measurement, or a fluorescence spectrophotometry.Examples of the method of measuring the β-D-galactosidase activity by aspectrophotometry (colorimetric method) include a method usingo-nitrophenyl-β-D-galactopyranoside. Examples of the method of measuringthe β-D-galactosidase activity by a luminescent measurement include amethod comprising reacting of β-D-galactosidase with its substrate, andmeasuring the luminescence of the reaction solution by a luminescenceintensity meter, luminescence multi-well plate reader, or the like.Examples of the substrate of β-D-galactosidase include Galacton-Plus(manufactured by Applied Biosystems), and analogs thereof. Examples ofthe method of measuring the β-D-galactosidase activity by a fluorescencespectrophotometry include a method comprising reacting ofβ-D-galactosidase with its substrate, and measuring the fluorescence ofthe reaction solution by a spectrofluorometer, fluorescence multi-wellplate reader, or the like. Examples of the substrate ofβ-D-galactosidase include 4-methylumbeliferyl-β-D-galactopyranoside.

In case the enzyme is luciferase, the luciferase activity can bemeasured, for example, by a luminescent measurement. Examples of themethod of measuring the luciferase activity by a luminescent measurementinclude a method comprising reacting of luciferase with its substrate,and measuring the luminescence of the reaction solution by aluminescence intensity meter, luminescence multi-well plate reader, orthe like. Examples of the substrate of luciferase include luciferin andcoelenterazine.

In case the labeling substance is those other than a fluorescentsubstance, a luminescent substance, a radioisotope, or an enzyme,detection can be carried out according to a method Comprising: allowinga labeled substance, in which a substance capable of specificallybinding to the labeling substance is labeled with a fluorescentsubstance, a luminescent substance, a radioisotope, an enzyme or thelike, to bind with the labeling substance constituting the labeledantibody or the labeled competitive substance of the immune complex;making the measurement by using the fluorescent substance, theluminescent substance, the radioisotope, or the enzyme, which label thesubstance capable of binding to the labeling substance, as describedabove. Examples of the substance capable of specifically binding to thelabeling substance include an antibody capable of specifically bindingto the labeling substance, avidin or streptavidin which are thesubstances capable of specifically binding to biotin (the labelingsubstance). Furthermore, detection can be carried out according to amethod comprising: allowing a substance capable of specifically bindingto the labeling substance, such as an antibody capable of specificallybinding to the labeling substance, and avidin or streptavidin, to bindwith labeling substance of the immune complex; then allowing a labeledantibody to bind with the labeling substance, wherein the labeledantibody is formed by labeling an antibody capable of binding to thesubstance which is capable of specifically binding to the labelingsubstance (examples of the antibody include an antibody capable ofspecifically binding to a constant region of an antibody, and anantibody capable of specifically binding to avidin or streptavidin) witha fluorescent substance, a luminescent substance, a radioisotope, anenzyme or the like; and making the measurement by using the fluorescentsubstance, the luminescent substance, the radioisotope, or the enzyme,as described above.

The antibody used in such detection, the antibody capable ofspecifically binding to avidin or streptavidin or the labelingsubstance, the antibody capable of specifically binding to a constantregion of an antibody, the antibody capable of specifically binding toavidin or streptavidin may be polyclonal or monoclonal antibody, orantibody fragments in which Fc portion has been removed, such as Fab,F(ab′)₂ obtained by pepsin treatment, and Fab′ obtained by pepsintreatment and reduction treatment.

(13) Determination of the Component to be Measured

For determination of the component to be measured, it is necessary tomake a calibration curve that shows the relationship between theconcentrations of the component and the measured values (amount ofinformation originated from label) using a standard substance, i.e.,solutions of the component with known concentrations. The concentrationof the component can be determined as follows: making the calibrationcurve; carrying out the measurement using the sample; and correlatingthe measured values obtained with the calibration curve produced inadvance.

(14) Aqueous Medium and Other Coexisting Substances

Examples of the aqueous medium used in the method of immunoassaying ofthe present invention include a deionized water, a distilled water, anda buffer, and a buffer is preferred. A buffer agent used for preparingbuffer is not particularly limited as long as it has buffering ability.Examples of the buffer include a buffer with pH 1 to 11, such as lactatebuffer, citrate buffer, acetate buffer, succinate buffer, phthalatebuffer, phosphate buffer, triethanolamine buffer, diethanolamine buffer,lysine buffer, barbiturate buffer, imidazole buffer, malate buffer,oxalate buffer, glycine buffer, borate buffer, carbonate buffer, glycinebuffer, or Good's buffer.

Examples of the Good's buffer include 2-morpholinoethanesulfonic acid(MES) buffer, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane(Bis-Tris) buffer, tris(hydroxymethyl)aminomethane (Tris) buffer,N-(2-acetoamido)imino diacetic acid (ADA) buffer,piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES) buffer,2-[N-(2-acetamido)amino]ethanesulfonic acid (ACES) buffer,3-morpholino-2-hydroxypropanesulfonic acid (MOPSO) buffer,2-[N,N-bis(2-hydroxyethyl)amino]ethanesulfonic acid (BES) buffer,3-morpholinopropanesulfonic acid (MOPS) buffer,2-{N-[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid (TES) buffer,N-(2-hydroxyethyl)-N′-(2-sulfoethyl)piperazine (HEPES) buffer,3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (DIPSO)buffer, 2-hydroxy-3-{[N-tris(hydroxymethyl)methyl]amino}propanesulfonicacid (TAPSO) buffer, piperazine-N,N′-bis(2-hydroxypropane-3-sulfonicacid) (POPSO) buffer,N-(2-hydroxyethyl)-N′-(2-hydroxy-3-sulfopropyl)piperazine (HEPPSO)buffer, N-(2-hydroxyethyl)-N′-(3-sulfopropyl)piperazine (EPPS) buffer,tricine[N-tris(hydroxymethyl)methylglycine] buffer,vicine[N,N-bis(2-hydroxyethyl)glycine] buffer,3-[N-tris(hydroxymethyl)methyl]aminopropanesulfonic acid (TAPS) buffer,2-(N-cyclohexylamino)ethanesulfonic acid (CHES) buffer,3-(N-cyclohexylamino)-2-hydroxypropanesulfonic acid (CAPSO) buffer, and3-(N-cyclohexylamino)propanesulfonic acid (CAPS) buffer.

The concentration of the buffer is not particularly limited as long asit is a concentration suitable for the measurement, and is preferably0.001 to 2.0 mol/L, more preferably 0.005 to 1.0 mol/L, and particularlypreferably 0.01 to 0.1 mol/L.

In the method of immunoassaying of the present invention, a metal ion, asalt, a sugar, a surfactant, an antiseptic agent, a protein, a proteinstabilizer, or such can be present together.

Examples of the metal ion include magnesium ion, manganese ion, and zincion.

Examples of the salt include sodium chloride and potassium chloride.

Examples of the sugar include mannitol and sorbitol.

Examples of the antiseptic agent include sodium azide, an antibiotic(streptomycin, penicillin, gentamicin, etc.), BioAce, Proclin 300, andProxel GXL.

Examples of the protein include bovine serum albumin (BSA), fetal bovineserum (FBS), casein, and BlockAce (manufactured by DainipponPharmaceutical Co., Ltd.).

Examples of the protein stabilizing agent include Peroxidase StabilizingBuffer (manufactured by DakoCytomation).

(15) Reagent of Immunoassay

The reagent of immunoassay of the present invention can be used for themethod of immunoassaying of the present invention, and comprises thebile acid derivative of (3) mentioned above, and if necessary, apolyoxyethylene nonionic surfactant. Examples of the reagent ofimmunoassay of the present invention include a reagent comprising acomponent selected from the group consisting of (i) to (iii) below, thebile acid derivative of (3), and if necessary, a polyoxyethylenenonionic surfactant:

-   (i) a first antibody capable of binding to the component and a    labeled second antibody capable of binding to the component;-   (ii) a labeled competitive substance and an antibody capable of    binding to both of the component and the competitive substance; and-   (iii) a competitive substance and a labeled antibody capable of    binding to both of the component and the competitive substance.

The form of the reagent of immunoassay of the present invention is notparticularly limited, as long as it is a form that enables the method ofimmunoassaying of the present invention. Examples of forms of thereagent include a liquid form, a freeze-dried form, or the like. In caseusing a freeze-dried form of reagent, it is used for the measurementafter being dissolved in the aforementioned aqueous medium or such.

As to the bile acid derivative, the antibody capable of binding to thecomponent, the competitive substance, the labeled antibody capable ofbinding to the component, and the labeled competitive substance used inthe reagent of immunoassay of the present invention, the aforementionedbile acid derivative, the aforementioned antibody capable of binding tothe component, the aforementioned competitive substance, theaforementioned labeled antibody capable of binding to the component, andthe aforementioned labeled competitive substance can be used,respectively. Furthermore, the reagent of immunoassay of the presentinvention can comprise, as necessary, the aforementioned aqueous medium,the aforementioned metal ion, the aforementioned salt, theaforementioned sugar, the aforementioned surfactant, the aforementionedantiseptic agent, the aforementioned protein, the aforementioned proteinstabilizer, or such.

Furthermore, the reagent of immunoassay of the present invention can bestored and distributed in the form of a kit. Examples of the kit includea kit composed of two reagents and a kit composed of three reagents, andthe components in each of the reagents constituting the kit can besuitably selected by those skilled in the art. For example, a solutionprepared by dissolving a bile acid derivative in an aqueous medium canbe comprised of the kit as a solution for dilution of a sample, whichcan be one of the reagents constituting the kit.

(16) Method of Suppressing an Interference of Hemoglobin in a Sample

The present invention provides a method of suppressing an interferenceof hemoglobin in the method of immunoassaying a component to be measuredin a sample containing hemoglobin. The interference of hemoglobin can besuppressed by co-existence of a bile acid derivative different from abile acid derivative that is inherently contained in the sample, in themethod of immunoassaying of the component to be measured. Co-existenceof a bile acid derivative different from a bile acid derivative that isinherently contained in the sample, suppresses an interference ofhemoglobin in the sample to give an accurate measurement.

Herein below, the present invention will be specifically described withreference to the Examples, which is not to be construed as limiting thescope of the present invention.

EXAMPLE 1

[1] Preparation of Anti-MxA protein monoclonal antibodies

A hybridoma cell line KM1124 (FERM BP-4729) which produces themonoclonal antibody KM1124 and a hybridoma cell line KM1135 (FERMBP-4731) which produces the monoclonal antibody KM1135 were individuallyintraperitoneally injected into pristane-treated 8-week old nude femalemice (Balb/c) at 5 to 20×10⁶ cells/animal. The hybridoma cell canceratedin ascites of mice after 10 to 21 days, and the ascitic fluids werecollected from the mice. The collected ascitic fluids were centrifugedat 3000 rpm for five minutes to remove the solid content, and thesupernatants were collected. Monoclonal antibodies were purified by thecaprylic acid precipitation method (Antibodies—A Laboratory Manual, ColdSpring Harbor Laboratory, 1988) from the supernatants, and themonoclonal antibodies KM1124 and KM1135 were obtained, respectively.

KM1124 is a mouse monoclonal antibody capable of binding to the epitopein residues 220 to 297 counting from the amino terminus of human MxAprotein, and KM1135 is a mouse monoclonal antibody capable of binding tothe epitope in residues 10 to 220 counting from the amino terminus ofhuman MxA protein.

[2] Preparation of Recombinant MxA Protein

With a human MxA protein expression vector pET14b-MxA (Nucleic AcidsRes., 32, 643-652, 2004) produced by inserting an NdeI-BamHI fragmentcontaining a cDNA encoding human MxA protein between NdeI and BamHI ofthe pET-14b vector (manufactured by Novagen, EMD Biosciences), wastransformed the Escherichia coli BL21 (DE3) pLysS strain. Thistransformant expresses an MxA protein to which an N-terminal His tag hasbeen added.

The obtained transformant was inoculated into 5 mL of LB mediumcontaining ampicillin, and the cells were cultured with shaking at 37°C. until the optical density at 600 nm (OD600) reached 0.5. This culturesolution was inoculated into 250 mL of LB medium containing ampicillin,and the cells were cultured with shaking at 37° C. until the opticaldensity at 600 nm reached 0.3 to 0.5. To this culture,isopropylthiogalactoside (IPTG) was added at a final concentration of0.4 mmol/L, and culturing was completed after two more hours ofsubsequent culture with shaking at 37° C. The culture solution wascentrifuged at 4° C. at 3000 rpm for ten minutes to collect thebacterial cells. The bacterial cells were stored at −80° C. until MxAprotein preparation.

Since MxA protein was present in the bacterial cells in the form ofinclusion bodies, the bacterial cells were thawed on ice, and 20 mL ofice-cooled binding buffer (5 mmol/L imidazole, 0.5 mol/L sodiumchloride, 20 mmol/L Tris-HCl, pH 7.9) was added to give the suspension.The bacterial cell suspension was subjected to five times of 30-secondultrasonic treatment to disrupt the cells, and then centrifuged at 4° C.at 4000 rpm for ten minutes. The supernatant was removed, and theprecipitate was suspended in 20 mL of added ice-cooled binding buffer.Similarly, ultrasonic treatment and centrifugation were again performed.The supernatant was removed, and 20 mL of the binding buffer containing6 mol/L urea was added to the precipitate to give the suspension. Aftera similar ultrasonic treatment, the mixture was left to stand on ice for30 minutes to dissolve the inclusion bodies, and then centrifuged at 4°C. at 10,000 rpm for 30 minutes. The supernatant was collected and thenfiltered through a 0.45-nm millipore filter.

To the obtained solution, 0.5 mL of Ni-NTA His•Bind Resin (manufacturedby Novagen, EMD Biosciences) was added, then the whole was mixed whilerotating at 4° C. for two hours, and the MxA protein was allowed to bindwith the resin via the His tag. This mixture was centrifuged at 4° C. at3000 rpm for two minutes to recover the resin. After adding 10 mL ofice-cooled binding buffer containing 6 mol/L urea to the resin, thewhole was centrifuged at 4° C. at 3000 rpm for two minutes to recoverthe resin. After repeating this washing operation, 10 mL of ice-cooledwashing buffer (6 mol/L urea, 60 mmol/L imidazole, 0.5 mol/L sodiumchloride, 20 mmol/L Tris-HCl, pH 7.9) was further added to the resin,and the whole was centrifuged at 4° C. at 3000 rpm for two minutes torecover the resin.

Ten mL of ice-cooled elution buffer (6 mol/L urea, 1 mol/L imidazole,0.5 mol/L sodium chloride, 20 mmol/L Tris-HCl, pH 7.9) was added to theresin, and the whole was mixed while rotating at 4° C. for two hours toelute the MxA protein from the resin. The mixture was subsequentlycentrifuged at 4° C. at 3000 rpm for two minutes, and the supernatantwas collected as the MxA protein solution.

[3] Preparation of Anti-MxA Protein Antibody-immobilized Plate

The anti-MxA protein monoclonal antibody KM1135 prepared in [1] wasdiluted with PBS to a concentration of 5 μg/mL, and the mixture wasdispensed in a 96-well microtiter plate (manufactured by Nalge NuncInternational) at 100 μL/well. After allowing the plate to stand forthree days, the supernatant was removed by suction, 25% BlockAce(manufactured by Dainippon Pharmaceutical Co., Ltd.) and 300 μL PBS weredispensed, and blocking was carried out at room temperature by allowingthe plate to stand overnight. After removing the blocking solution,washing was carried out using PBS. The plate after drying for three daysusing a vacuum dryer was used as the anti-MxA protein monoclonalantibody-immobilized plate.

[4] Preparation of Peroxidase-labeled Anti-MxA Protein Antibody

The anti-MxA protein monoclonal antibody KM1124 prepared in [1] wasallowed to bind with peroxidase (hereinafter, abbreviated as POD) by themaleimide method as described below to give a POD-labeled anti-MxAprotein antibody.

First, the solvent of a solution containing 2 mg of the anti-MxA proteinantibody KM1124 was substituted with 0.1 mol/L borate buffer (pH 8.0),and 0.086 mg of 2-iminothiolane hydrochloride (manufactured by PIERCE)was added. The mixture was stirred and the reaction was continued at 30°C. for 30 minutes after stirring. Using a Sephadex G25 (manufactured byAmersham Bioscience) column (1.5 cm diameter×30 cm) equilibrated with0.1 mol/L phosphate buffer (pH 6.0), unreacted 2-iminothiolane in thereaction solution was removed and sulfhydrylated KM1124 was collected.

Meanwhile, 2.5 mg of POD (manufactured by TOYOBO, peroxidase I-C), whichcorresponds to 5-times the amount of the anti-MxA protein antibody KM1124 in terms of molar ratio, was dissolved in 250 μL, of 0.1 mol/Lphosphate buffer (pH 7.0). After warming this solution at 30° C. forfive minutes, 0.72 mg of N-(6-maleimidocaproyloxy)succinimide (EMCS,manufactured by DOJINDO Laboratories) dissolved in N,N-dimethylformamide(manufactured by Nacalai Tesque) was added, and the mixture was stirredand the reaction was continued at 30° C. for 30 minutes. Using aSephadex G25 column (1.5 cm diameter×30 cm) equilibrated with 0.1 mol/Lphosphate buffer (pH 6.0), the reacted solution was subjected to gelfiltration to remove unreacted EMCS, and maleimidated POD was collected.

The above-obtained solution of sulfhydrylated anti-MxA protein antibodyKM1124 was mixed with the solution of maleimidated POD, and allowed toreact at 30° C. for one hour. The obtained labeled antibody was diluted800 times with a POD label diluent (liquid composition) buffer [50mmol/L Bis-Tris (manufactured by DOJINDO Laboratories), 0.1% BSA(manufactured by InterGen)].

[5] Preparation of Sample Diluents

Sample diluents of the following compositions were preparedindividually.

HEPES (pH 8.0) 0.1 mol/L Surfactant (type and concentration shown inTable 1) NaCl 1.5 mol/L BSA 0.1% Sodium azide 0.1%[6] Construction of an MxA Protein Assay System Using Sandwich ELISA

The MxA protein solution prepared in [2] mentioned above was dilutedusing the sample diluent prepared in [5] mentioned above to give thesolutions of the MxA protein at the concentrations of 0 (buffer only),3.2, 6.3, 12.5, 25, 50, 100, and 200 ng/mL, and these solutions wereused as the samples for measurement.

To the anti-MxA protein antibody-immobilized plate produced in [3]mentioned above was added 100 μL of the samples for measurement, andthen the mixture was incubated at room temperature for one hour to allowthe MxA protein in the samples for measurement to bind to the antibody.After removing the samples for measurement, a washing operation ofaddition of 400 μL of washing solution (PBS containing 0.05% Tween 20(manufactured by KANTO CHEMICAL)) followed by removal of the washingsolution was performed five times. Next, 100 μL of the POD-labeledanti-MxA protein antibody solution prepared in [4] was added, and thereaction was continued at room temperature for 30 minutes. The labeledantibody was removed, and a washing operation of addition of 400 μL ofwashing solution and its removal was performed five times. In the dark,100 μL of TMBlue (manufactured by Serological), which is a chromogenicsubstrate of POD containing 0.05% tetramethylbenzidine and hydrogenperoxide, was added and the reaction was continued at room temperaturefor ten minutes. The reaction was stopped by adding 100 μL of 0.5 mol/Lsulfuric acid and incubating at room temperature for ten minutes. Theabsorbance at wavelength 450 nm was measured using a plate reader. Theresults showed that the absorbance increased as the concentration of MxAprotein in the samples for measurement increased, and it proved that theMxA protein could be measured.

Actually, in case of the measurement of the concentration of MxA proteinin the blood, a calibration curve obtained in this manner is used todetermine the MxA protein in the blood.

[7] An Addition and Recovery Test of MxA Protein Using Blood

Blood collected from two MxA protein-positive patients found to havevirus infection and from three healthy individuals using EDTA•2Na bloodcollection tubes was used.

Samples obtained by ten-fold dilution of this blood using the samplediluent of [5] were subjected to the measurement of the MxA proteinconcentration in each sample according to the method [6], and thedetermined concentrations were defined as the MxA protein concentrationin a sample without added MxA protein (hereinafter, abbreviated as A).

Next, samples prepared by adding 1 part of the 500 ng/mL MxA proteinsolution prepared in [2] mentioned above to 9 parts of these samplesolutions were subjected to the measurement of the MxA proteinconcentration in each sample according to the method [6], and thedetermined concentrations were defined as MxA protein concentration inMxA protein-added samples (hereinafter, abbreviated as B). The MxAprotein recovery rates (%) was calculated from the equation below:MxA Protein Recovery Rate (%)=(B−A)/50*100  (Equation 1)

Theoretically, the MxA protein recovery rate (%) becomes 100% when aninterference of hemoglobin is completely suppressed, and its value willdecrease as the interference of hemoglobin appear.

COMPARATIVE EXAMPLE 1

The addition and recovery test was performed by a similar method toExample 1, except that a sample diluent lacking the surfactant from thecomposition of [5] in Example 1 was used as the sample diluent.

COMPARATIVE EXAMPLE 2

The addition and recovery test was performed by a similar method toExample 1, in which 0.2% Nonidet P40 was used as the surfactant in thecomposition of [5] in Example 1.

TABLE 1 Concentration MxA protein recovery rate (%) Surfactant (%)Positive 1 Positive 2 Negative 1 Negative 2 Negative 3 Average CHAPS 4.988.9 96.6 97.5 93.8 88.2 93.0 CHAPSO 5.0 76.3 92.1 100.0 91.4 86.9 89.3BIGCHAP 2.5 76.7 91.8 103.3 86.6 88.2 89.3 deoxy-BIGCHAP 1.2 70.6 82.982.8 81.4 72.5 78.0 Surfactant-free 0 70.2 67.0 66.9 75.7 72.8 70.5(Comparative Example 1) Nonidet P-40 0.2 67.9 75.7 82.6 68.2 67.9 72.5(Comparative Example 2)

Table 1 shows that addition of a bile acid derivative in themeasurements increases the level of MxA protein recovery rate, whichindicates that an interference of hemoglobin is suppressed.

EXAMPLE 2

The sensitivity due to the addition of Nonidet P-40 to the samplediluent composition described in [5] above containing 4.9% CHAPS wasexamined. Nonidet P-40 was added to the sample diluent of [5] describedabove at 0%, 0.2%, 1%, and 1.4%, and solutions of the MxA proteinprepared in the above-mentioned [2] were prepared at the concentrationsof 0 (buffer only), 3.2, 6.3, 12.5, 25, 50, 100, and 200 ng/mL, and theabsorbances (Abs) were measured by the method described in [6] inExample 1. The results are shown in Table 2.

TABLE 2 MxA protein Concentration of Nonidet P-40 added (%) (ng/mL) 0.00.2 1.0 1.4 0 0.022 0.022 0.022 0.022 3 0.038 0.041 0.055 0.060 6 0.0550.061 0.087 0.096 12 0.082 0.093 0.140 0.157 24 0.139 0.158 0.244 0.27348 0.243 0.274 0.424 0.477 96 0.433 0.496 0.761 0.853 192 0.810 0.9151.387 1.528

Table 2 shows that the absorbance increases to give an increasedsensitivity, depending on the amount of Nonidet P-40.

EXAMPLE 3

Blood collected from two MxA protein-positive patients found to havevirus infection and from three healthy individuals using EDTA•2Na bloodcollection tubes was used as samples.

The MxA protein recovery rate was determined in a similar manner as inExample 1, except that a sample diluent containing 4.9% CHAPS, a samplediluent containing 4.9% CHAPS and 1.4% Nonidet P-40 were used as thesample diluent. The results are shown in Table 3.

TABLE 3 MxA protein recovery rate (%) Surfactant Positive 3 Positive 4Negative 4 Negative 5 Negative 6 Average CHAPS 93.9 89.3 96.1 97.5 98.395.0 CHAPS + 90.1 83.8 96.1 100.5 104.2 94.9 Nonidet P-40

Table 3 shows that the recovery rate does not decrease even when NonidetP-40 is added.

INDUSTRIAL APPLICABILITY

The present invention provides a method of immunoassaying a component tobe measured in a sample containing hemoglobin, which effects asuppression of an interference of hemoglobin and is useful for clinicaldiagnosis.

1. A method of immunoassaying a component to be measured in a samplecontaining hemoglobin, which comprises: (i) reacting said component tobe measured with an antibody capable of binding to the component in thepresence of a bile acid derivative different from a bile acid derivativethat is inherently contained in the sample to form an immune complex;and (ii) determining the amount of the component in the immune complex,wherein the component to be measured is a substance contained withincells or a protein induced within cells by cytokines wherein the bileacid derivative has amphoteric surfactant function or nonionicsurfactant function.
 2. The method according to claim 1, which comprisesreacting said component to be measured in a sample containing hemoglobinwith an antibody capable of binding to the component, further in thepresence of a polyoxyethylene nonionic surfactant.
 3. The methodaccording to claim 1 or 2, wherein the method of immunoassay is asandwich method or a competition method.
 4. The method according toclaim 1 or 2, wherein reacting the component with an antibody capable ofbinding to the component comprises: (1) reacting the component with afirst antibody capable of binding to the component and a labeled secondantibody capable of binding to the component, (2) reacting the componentwith a labeled competitive substance and an antibody capable of bindingto both of the component and the competitive substance, or (3) reactingthe component with a competitive substance and a labeled antibodycapable of binding to both of the component and the competitivesubstance.
 5. The method according to claim 1 or 2, wherein the bileacid derivative different from a bile acid derivative that is inherentlycontained in the sample is a bile acid derivative having amphotericsurfactant function.
 6. The method of claim 1 or 2, wherein the bileacid derivative different from a bile acid derivative that is inherentlycontained in the sample is a bile acid derivative having nonionicsurfactant function.
 7. The method of claim 5, wherein the bile acidderivative having amphoteric surfactant function is3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate or3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxypropanesulfonate.
 8. Themethod according to claim 6, wherein the bile acid derivative havingnonionic surfactant function is N,N-bis(3-D-gluconamidopropyl)cholamideor N,N-bis(3-D-gluconamidopropyl)deoxycholamide.
 9. The method accordingto claim 2, wherein the polyoxyethylene nonionic surfactant ispolyoxyethylene alkylphenyl ether.
 10. The method according to claim 1or 2, wherein the sample is whole blood.
 11. The method according toclaim 1 or 2, wherein the component to be measured is MxA protein.